1. Field of the Invention
The invention is generally directed to the automation of biological sample processing, and is specifically directed to a method and apparatus for automated staining of biological samples using a low-volume tangential fluid approach.
2. Description of Related Art
Staining of biopsied tissue or cellular preparations for morphological visualization is an ancient art by modern standards that goes back over one hundred years. Recently, efforts have been made to automate the procedure of applying different types of chemical stains and biological conjugate molecules to tissue sections. Instruments that have been designed for this purpose include the Ventana Medical Systems' line of dual carousel-based instruments such as the 320/ES®, NexES®, BENCHMARK®, and the BENCHMARK® XT. Patents that describe these systems include U.S. Pat. Nos. 5,595,707, 5,654,199, 6,093,574, and 6,296,809, all of which are incorporated herein by reference in their entirety. Another type of automated stainer is the TechMate® line of stainers, described in U.S. Pat. Nos. 5,355,439 and 5,737,499, both of which are incorporated herein by reference in their entireties.
The rate of Immunohistochemical and in situ hybridization staining of sectioned fixed tissue on a microscope slide is limited by the speed at which the conjugating biomolecules can diffuse into the fixed tissue from an aqueous solution placed in direct contact with the tissue section. Typically, tissue is “fixed” immediately after excision by placing it in a 10% solution of formaldehyde, which preserves the tissue from autocatalytic destruction by cross-linking much of the protein via methylene bridges. This cross-linked tissue presents many additional barriers to diffusion including the lipid bilayer membranes that enclose individual cells and organelles, and the aforementioned effects of cross-linking that the fixation process generates. The conjugate biomolecules (antibody or DNA probe molecules) are relatively large, ranging in size from a few kilo Daltons to several hundred kiloDaltons, which constrains them to diffuse slowly into solid tissue with typical times for sufficient diffusion being in the range of several minutes to a few hours. Typical incubation conditions are thirty minutes at 37 degrees centigrade.
The diffusion rate is driven by a concentration gradient so the rate can be increased by increasing the concentration of the conjugate in the reagent. However, this has two detrimental effects. First, the conjugates are often very expensive, so increasing their concentration is wasteful and often not economically viable. Second, the excessive amount of conjugate that is driven into the tissue, when high concentrations are used, is entrapped in the tissue, and is difficult to rinse out and causes high levels of non-specific background staining. Non-specific staining is just noise. In order to reduce the noise and increase the signal of specific staining, current practice dictates using low concentrations of conjugate with long incubation times to allow the conjugate to find and bind to only the specific sites.
Automation of the previously manual processes of diffusion-driven staining has only increased these issues due to the necessarily larger pools of reagents. Present histology staining instruments use relatively large volumes of reagent (100 μl) in a puddle of typically 300 μl of buffer, as disclosed in issued U.S. Pat. Nos. 6,352,861, 6,296,809 and others. This produces a rather low concentration of the conjugate reagent in the puddle that resides over the tissue. Present instruments mix the reagent by alternating tangential air jets onto an overlaying oil layer that rotates and counterrotates when contacted by the alternating air jets, thereby imparting motion into the underlying aqueous puddle. This mixing is slow and not particularly vigorous, and creates evaporation issues that must be countered. The oil layer minimizes evaporation of the aqueous puddle by covering it with a layer of low vapor-pressure oil. Finally, present instruments use large volumes of rinse liquid to physically displace the reagent's large puddles of low concentration reagents which are covered with oil. This rinsing method produces large volumes of waste liquid which may be classified as hazardous waste, and in any event can physically disrupt the tissue by the vigorous washing action.
There continues to be a need for faster introduction of biomolecules into tissue sections for quicker processing and lower-volume reagent usage.